This invention relates to hydrogels for biomedical applications.
Chitin is an abundant, naturally occurring polymer of N-acetylglucosamine which is present in fungi and in the exoskeletons of crustaceans and insects. When chitin is treated with strong base such as sodium hydroxide, deacetylation occurs to produce the polymer called chitosan. Such chitosan is commonly 80-90% deacetylated. Chitosan is soluble in aqueous acid, but insoluble in water. 
Chitosan is of increasing interest in drug delivery. It is known, for example, to enhance transport of hydrophilic drugs such as peptides and proteins across the intestinal epithelial barrier (N. G. Schipper, K. M. Varnum, and P. Artursson, Pharm. Res., 13, 1686-1692, 1996). It has also reported to be useful in both colon delivery (H. Tozaki, et. al, J. Pharm, Sci., 86, 1016-1021, 1997) and nasal delivery of insulin (R. Soane, et. al, Proc. 25th International Symp. on Controlled Release of Bioactive Materials, 1998). Chitosan is also of current interest as a carrier in gene delivery (MacLaughlin, et. al, J. Controlled Release, 56, 259-272, 1998).
Hydrogels, which are cross-linked polymers that swell in water, have potential as drug delivery vehicles. However, many of the hydrogels that have been reported have drawbacks and disadvantages that detract from their use as either drug delivery vehicles or in other biomedical applications. Some cross-linking agents are considered to be toxic and could be problematic if released from the hydrogel in vivo. Some gels are prepared with multiple constituents that can unnecessarily complicate of the preparation of the gels. Some gels are not as stable as desired when used in vivo. It would be desirable to develop new hydrogels that reduce or eliminate some of these drawbacks and disadvantages.
The invention provides cross-linked polymers of chitosan or alkoxy poly(alkylene oxide) conjugates of chitosan with multifunctionalized poly(alkylene oxide), and methods for their preparation and use. The poly(alkylene oxides) used in the invention are typically poly(ethylene glycols) (xe2x80x9cPEGsxe2x80x9d), and the discussion below is based on PEG, although it should be recognized that the invention includes other poly(alkylene oxides), including copolymers of ethylene oxide and propylene oxide. These cross-linked structures produce hydrogels in the presence of water that can be useful for, among other things, administering therapeutic agents to humans and other mammals, for the prevention of surgical adhesions, as surgical sealants, as wound dressings, and for the treatment of scars.
In one embodiment, the gels are hydrolytically stable and thus remain intact in vivo for an extended period. In another embodiment, the gel is degradable and provides a water-soluble form of chitosan. The degradable gel can be used for medical imaging applications, in addition to the uses listed above, in which an imaging agent is delivered in vivo.
No cross-linking agents other than chitosan and poly(ethylene glycol) are typically used in the preparation of the hydrogel and the hydrogel can be prepared from chitosan and a single multifunctionalized poly(ethylene glycol). The hydrogel can also be prepared from a chitosan and poly(ethylene glycol) conjugate cross-linked with a multifunctionalized poly(ethylene glycol).
In certain applications, it is advantageous that the chitosan used in forming the cross-linked polymer be present in a form that is water-soluble at or near neutral pH. This invention thus further provides for use of covalently attached monoalkoxy PEG, such as methoxy PEG, on some of the amino groups of the chitosan so that a water-soluble alkoxyPEGylated chitosan can be used for cross-linking with multifunctionalized PEG. The chitosan-PEG conjugate and the cross-linking PEG reagent can thus, in one embodiment, be delivered in solution from separate chambers to form a hydrogel upon mixing of the two solution streams.
Monofunctional PEG moieties, such as alkoxy PEG derivatives, may be attached to chitosan by a variety of methods. For example, PEG can be attached to amino groups on chitosan using an activated PEG carboxylic acid. Such activated PEG carboxylic acids may include acid chlorides, N-succinimidyl esters, 1-hydroxybenzotriazole esters and related activated PEG carboxylic acids. PEG may also be attached to amino groups of the chitosan by a carbonate (urethane) linkage by reaction with PEG chloroformate or an activated PEG carbonate, such as an N-succinimidyl ester or a 1-benzotriazole ester of a PEG carbonate. In another embodiment, a urea linkage may be formed by reaction of chitosan amino groups with an alkoxy PEG isocyanate. Alkoxy PEG may also be attached to chitosan amine groups by reductive amination using sodium cyanoborohydride and a PEG aldehyde, such as mPEG acetaldehyde or mPEG propionaldehyde, or the corresponding aldehyde hydrates and sodium cyanoborohydride. Similar linkages can be formed by reaction of chitosan with PEG activated by appropriate leaving groups such as halide, tosylate, mesylate, or tresylate.
This invention provides methods for cross-linking multifunctional PEG by reaction with amino groups on alkoxy PEG-chitosan conjugates or on chitosan. Such PEG may be difunctional or it may have a greater number of functional groups including, but not limited to, those PEG derivatives prepared from 3-arm, 4-arm, 8-arm or more PEG. Useful activating groups on the termini of the multifunctional PEG are the same as those described above for attaching alkoxy PEG to chitosan. Included are activated derivatives of PEG carboxylic acids, such as N-hydroxysuccinimidyl esters or 1-benzotriazolyl esters. Also included are PEG isocyanates, PEG aldehydes or aldehyde hydrates, and PEG tosylates, mesylates; or tresylates. Cross-linking occurs with the formation of amide, carbamate, or amine linkages to chitosan or an alkoxy PEG-chitosan conjugate.
In certain applications it is advantageous to utilize a hydrogel that breaks down into smaller, water-soluble molecules that can be more readily eliminated from the body. This invention provides for functional groups in the backbone of the cross-linking PEG that can be hydrolyzed at ambient pH or by enzymatic catalysis, or can degrade photochemically. Functional groups that are subject to hydrolysis include, but are not limited to, carboxylate esters, phosphates, sulfates, orthoesters, acetals, certain amides, and certain carbamates. Hydrolytic degradation of the cross-linking PEG moieties results in conversion of the cross-linked chitosan to chitosan that is covalently linked to a PEG, moiety.
In yet another embodiment, the cross-linking PEG can be prepared with a backbone group that is subject to photolytic cleavage. Cinnamylidine esters, for example, dimerize at 313 nm and reversibly cleave at 254 nm. Thus if a PEG having a terminal cinnamylidine ester is linked to chitosan, cross-linking will occur at 313 nm and the process can be reversed at 254 nm.
Biologically active molecules, such as small drug molecules, proteins, peptides, lipids, DNA, carbohydrates, imaging agents, or oligonucleotides, can be physically entrapped in the gel and delivered by diffusion from the hydrogel. Biologically active molecules may also be covalently bound to the amino groups or to the hydroxyl groups of the chitosan moiety of the hydrogel or to a poly(ethylene glycol) moiety.